A Novel Platform to Generate Human Induced Pluripotent Stem Cells for Developing Intestinal Disease Models

生成人类诱导多能干细胞用于开发肠道疾病模型的新平台

• 批准号: 9610915
• 负责人: Nicole M Smiddy
• 金额: $ 3.61万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9610915
• 关键词: Address; Area; Back; Biological Assay; Biological Markers; Biopsy; Cell Culture Techniques; Cell Differentiation process; Cell Proliferation; Cell Separation; Cells; Collection; Colorectal; Development; Digestive System Disorders; Disease model; Elements; Endoderm; Ensure; Erythroblasts; Exhibits; Flow Cytometry; Genes; Genomics; Human; Immunoassay; Immunofluorescence Immunologic; In Vitro; Individual; Inflammatory; Intestinal Diseases; Intestines; Link; Magnetism; Manuals; Methods; Modeling; Morphology; Organoids; Pattern; Population; Prevalence; Principal Component Analysis; Reagent; Reporting; Research; Research Personnel; Sampling; Site; Somatic Cell; Sorting - Cell Movement; Surface; System; Technology; Testing; Time; Work; base; cancer cell; cell type; cellular transduction; design; experimental study; fluorescence imaging; high throughput technology; induced pluripotent stem cell; insight; novel; peripheral blood; pluripotency; response; self-renewal; stem-like cell

7. Project Summary Over the past 25 years, the prevalence of digestive diseases worldwide has increased by ~25%.1 Within the past year, intestinal organoids derived from hiPSCs have resulted in pioneering in vitro studies of colorectal cancer2, inflammatory bowel disease3, and intestinal response to infection8. Because the conversion efficiency of somatic cells into hiPSCs is low (~0.01-1%10), it is essential that the hiPSCs be purified quickly and efficiently after reprogramming to allow for rapid and successfully development of hiPSC-derived intestinal models. Current purification methods range from non-specific and time-intensive morphological analysis12 to flow cytometry sorting based on surface markers which imposes high shear forces onto hiPSCs often resulting in unwanted differentiation15. To streamline the application of hiPSCs in intestinal disease models, a universally simple, unbiased, and gentle technology for identifying and isolating hiPSCs must be developed. I aim to address the limitations of existing hiPSC sorting methods by creating an automated technology based upon microraft arrays. Microraft arrays are microwell arrays with each individual microwell containing a magnetic releasable cell culture element (“microraft”).23 Each microraft cell carrier can be gently released from the array and easily collected with a magnet for assays of their adhered cells. The proposed platform will advance microraft arrays to allow for every micro-colony of transduced erythroblasts cultured on the array to be biopsied and each colony fragment collected into a designated microwell within a replicate well array. Within the collection wells, all colony fragments are assayed simultaneously via cell-lethal immunoassays for key pluripotency biomarkers: OCT4, SOX2, TRA-1-60, and SSEA4. By mapping the assayed fragments back to the living original colony on the microraft array, the original colonies that are determined to express distinct markers of hiPSCs can be resampled, and the cells validated via genomic and differentiation assays. Studies made possible using the platform will result in new insight into the relationship between the primary hiPSC colonies’ proliferation rate and biomarker expression, as well as the established hiPSC colonies’ potential for self-renewal, trilineage differentiation, and development into intestinal cells. It is important to note that while this proposal limits its scope to isolating hiPSCs, the microraft array platform can easily be adapted to enrich for essentially any rare cell population with a distinct intracellular or surface biomarker, such as sub- populations of cancer cells and successfully gene-edited or differentiated cells. The platform will also provide researchers with a gentle, biomarker-based method for sorting sensitive cell types and performing higher throughput cell sorts based on intracellular markers.

7.项目总结 在过去的25年里，全球消化系统疾病的患病率增加了约25%。 在过去的一年里，来自HiPSCs的肠道有机物质导致了对结直肠的开创性的体外研究 癌基因2、炎症性肠病3、肠道对感染的反应8。因为转换效率 体细胞转化为HiPSCs的比例很低(~0.01-1%10)，因此有必要迅速纯化并 重新编程后高效，以支持HiPSC来源的肠道的快速和成功发展 模特们。目前的纯化方法从非特异性和耗时的形态分析12到 基于表面标记的流式细胞术分选，这种分选对HIPSC施加高剪切力，通常导致 在不想要的差别中15。为了简化HiPSCs在肠道疾病模型中的应用，一个普遍的 必须开发简单、公正和温和的技术来鉴定和分离HiPSCs。 我的目标是通过创建基于以下内容的自动化技术来解决现有HiPSC分类方法的局限性 在微型后方阵列上。微孔阵列是微孔阵列，每个单独的微孔包含 磁性可释放细胞培养元件(“微载体”)。23每个微载体可轻轻地从 该阵列可以很容易地用磁铁收集，以进行贴壁细胞的检测。拟议的平台将 先进的微阵列，以允许在阵列上培养的每一个转导的红细胞微集落 活组织检查并将每个菌落片段收集到复制井阵列内的指定微孔中。在 收集井中，所有菌落片段通过细胞致死免疫分析同时检测 多能生物标志物：Oct4、SOX2、TRA-1-60和SSEA4。通过将检测到的片段映射回 活在微阵列上的原始菌落，决心表达不同的原始菌落 可以重新采样HiPSCs的标记，并通过基因组和分化分析验证细胞。 使用该平台进行的研究将使人们对小学和小学之间的关系有新的认识 HIPSC集落的增殖率和生物标志物的表达以及已建立的HiPSC集落的 具有自我更新、三倍体分化和发育为肠道细胞的潜力。值得注意的是， 虽然这项提议将其范围限制为分离HiPSC，但微阵列平台可以很容易地进行调整 用不同的细胞内或表面生物标记物来丰富基本上任何稀有细胞群，例如亚细胞 癌细胞群体和成功进行基因编辑或分化的细胞。该平台还将提供 研究人员使用温和的、基于生物标记物的方法对敏感细胞类型进行分类，并获得更高的性能 吞吐量细胞根据细胞内标记进行排序。